The overall goal of the proposal is to determine the mechanisms by which effective cytotoxic T lymphocyte (CTL) response can be elicited against highly conserved internal virion proteins of HIV-1 gag and pol. Toward this goal, a novel development will be the construction of DNA expression vectors, which will enable the applicants to express gag or other HIV-1 structural proteins in a rev and RRE independent and species independent fashion. Using this new method, the applicants plan to systematically evaluate the expression of, and immune response to these different forms of HIV-1 gag, as well as to maximize the induction of CTL response against gag in mice. There will be two specific aims: (1) to construct in vitro different forms of HIV-1 gag expression vectors in a variety of cells. Using a novel strategy, the investigators intend to generate HIV-1 gag expression vector in the absence of rev and RRE. If successful, this will permit the expression of HIV-1 gag molecules in mouse cells, a task which has proven to be difficult even with rev and RRE. Various forms of HIV-1 gag expression vectors, which are targeted to different intracellular and extracellular compartments for antigen processing and presentation, will be constructed and evaluated in vitro. The second specific aim is to characterize the immune response of mice, especially CTL, with vectors expressing different forms of HIVI gag. Furthermore, the applicants will evaluate the ability of different forms of HIV-1 gag DNA vaccine to induce and maximize CTL response in mice. These different forms of gag include: (a) wild type gag that assembles into particles which are released from cells efficiently; (b) mutant gag forms that fail to assemble into particles and remain in the cytoplasm; (c) mutant gag forms that are rapidly degraded intracellularly; (d) secreted but non-particle forms of gag; (e) cell surface anchored forms of gag; and f) chimeric gag that is targeted specifically to lysosome/endosome for MHC class II presentation. The proposed studies are conceptualized as the necessary ground work for subsequent evaluations in non-human primate models in which virus challenging experiments can be performed.